Etiket Arşivleri: Laboratory

Laboratory‎ > ‎Seafood ( Hakan MAVİŞ )

FE 421 FOOD MICROBIOLOGY LABORATORY

Name of student : M. Hakan MAVİŞ

Group : B – 2

Name of experiment : Seafood

PURPOSE:

The purpose of this experiment was to investigate microbiological affectivities of fish.

THEORY:

International competitiveness requires optimal productivity, quality and value, and the development of new products from traditional raw materials, underutilized species and waste streams. The productivity and competitiveness of seafood processing depends not only on the sources and costs of raw materials, but also on other costly resources: energy, water, labor and waterfront space. Energy equipment for thermal operations (refrigeration, cooking and retorting) is tremendous, yet opportunities exist for conservation through energy and water audits and demonstrating new technologies at processing plants. Solid waste disposal is a mounting problem for the industry as coastal populations and environmental sensitivities increase. This problem by developing enzymatic and microbial methods of hydrolysate manufacture for feed and fertilizer production, and improving manufacturing methods and uses of dried meals.

Seafood is among the most expensive items in the American diet due to the high costs of catching, transporting, processing and storing this delicate commodity. Although profit margins are small, improved post-harvest technologies offer opportunities to increase product quality and profits. Seafood muscle tissues are the most valuable component of seafood products—they have many desirable properties due to their water- and fat-binding traits, which can be enhanced by non-seafood additives and novel processing techniques. Ready-to-cook and ready-to-eat seafood products require processing and storage that can reduce product quality. A better understanding of the chemical and physical properties of seafood muscle components could minimize these effects. Many fish species are not widely consumed for food because they degrade rapidly. Improved storage and processing techniques would help; but because fish and shellfish are highly variable in their physiology, their properties need to be studied by species. New enzymes, enzyme inhibitors and other “active” proteins, such as antifreeze proteins, could be isolated from seafood sources and used to add value to other seafood.

MATERIALS:

  • Tubes

  • Fish

  • PCA (plate count agar)

  • Bunsen burner

  • Spreader

  • Sterile water

  • Alcohol

  • Etuv

  • Pipette

  • Test tube rack

PROCEDURE:

Firstly; a sterile swab was taken and it was plunged into 0,1 % peptone water for soaking then swab was spread on surface of fish about 10 cm2, swab was put into 10 ml 0,1 % peptone water after breaking tip off of swab then. Swab and peptone water were shaken then 0,5 ml sample was taken from tube containing swab and peptone water and inoculated to PCA and incubated at 37 oC for 24 hours.

Secondly; coliform test was applied on fish. 0,5 ml sample was taken and inoculated to violet red bile agar (VRBA) and then incubated at 37 oC for 48 hours. Finally; number of microorganisms was calculated in 1 cm2.

RESULTS and CALCULATIONS:

Total count

Coliform test

Group 1

216

11

Group 2

177

7

Group 3

244

15

Group 4

152

40

Group 5

202

24

Group 6

200

108

Group 7

147

17

Group 8

90

7

Group 9

110

5

These values were in 10cm2 and for 1cm2:

# of m/o’ s = 216*1 cm2 / 10cm2 = 22

# of m/o’ s = 177*1 cm2 / 10cm2 = 18

# of m/o’ s = 244*1 cm2 / 10cm2 = 24

# of m/o’ s = 152*1 cm2 / 10cm2 = 15

# of m/o’ s = 202*1 cm2 / 10cm2 = 20

# of m/o’ s = 200*1 cm2 / 10cm2 = 20

# of m/o’ s = 147*1 cm2 / 10cm2 = 15

# of m/o’ s =   90*1 cm2 / 10cm2 = 9

# of m/o’ s = 110*1 cm2 / 10cm2 = 11

Coliform test:

# of m/o’ s =   11*1 cm2 / 10cm2 = 1

# of m/o’ s =     7*1 cm2 / 10cm2 = 1

# of m/o’ s =   15*1 cm2 / 10cm2 = 2

# of m/o’ s =   40*1 cm2 / 10cm2 = 4

# of m/o’ s =   24*1 cm2 / 10cm2 = 2

# of m/o’ s = 108*1 cm2 / 10cm2 = 11

# of m/o’ s =   17*1 cm2 / 10cm2 = 2

# of m/o’ s =     7*1 cm2 / 10cm2 = 1

# of m/o’ s =     5*1 cm2 / 10cm2 = 1

DISCUSSION:

In this experiment we studied the seafood and for this we used a fish, and at 10cm2 number of microorganisms were examined. In order to analyze we used swab. Swab is a sterile loop. Swab was spread the surface of fish then heat part or cotton part was broken down so as to not touch our hand, because on our hand some microorganisms may be and these microorganisms with swab together can be put in 10 ml sterile water. If it is so, our results can be false so we must care when this process was applied.

Living fish carries gram negative psychrotropic bacteria on their surface and also fresh fish carries 102 or 103 bacteria per 1cm2 on surface, also our result shows in results and calculation part. Stale fish can be to include more microorganisms and these microorganisms can harm to human health.

Laboratory‎ > ‎Determination of Proteins

PURPOSE

To gain knowledge about the methods for protein determination and determination of protein content of a solution by using Biuret method.

PROCEDURE

A series solution of Bovine Serum Albumin (BSA) was prepared with different concentrations from 0 to 10 mg/ml. These concentrations were prepared by using stock solution of 10 mg/ml BSA and distilled water. Firstly a blank solution was prepared by using 0 ml BSA and 1 ml distilled water to be used for calibration of spectrophotometer. Then 0,8 ml distilled water was added to 0,2 ml BSA and 2 mg/ml solution was obtained. 0,7 ml distilled water was added to 0,3 ml BSA and 3 mg/ml solution was obtained. 0,5 ml distilled water was added to 0,5 ml BSA and 5 mg/ml solution was obtained. 0,2 ml distilled water was added to 0,8 ml BSA and 8 mg/ml solution was obtained. Finally no distilled water was added to 1,0 ml BSA and 10 mg/ml solution was obtained. After that 4 ml Biuret reagent was added to these test tubes. After waiting for 30 min. the absorbance of each tube was read at 540 nm and the results were recorded. By using these values a standard curve was plotted as concentration (mg/ml) vs absorbance (A540) and the concentration of our unknown protein solution was found.

‎Laboratory‎ > ‎Dialysis

In this experiment we observed the dialysis. The principle of dialysis is that the small molecules can penetrate through the pores of semi-permeable membrane toward the buffer solution while large molecules cannot. By using this method we separated glucose from starch and cystein from Bovine Serum Albumin (BSA). So the small molecules (glucose and cystein) were collected in buffer side and large molecules (starch and BSA) were kept in the bag. Then we tested the buffer solutions and the solutions in the bag with the reagents to decide if the separation process was completed successfully or not.

The samples taken from the buffer solution and the solution inside of the bag of starch- glucose mixture was tested with Lugol’s solution and Benedict reagent. Lugol’s solution is used to observe the presence of starch since the colour changes from yellow to blue-black in the presence of starch. The result showed that there was starch in the bag but there was not any in the buffer. This result was expected. Then Benedict reagent was used which changes colour from blue to red in the presence of glucose. The result showed that there was glucose in the buffer but there was not any in the bag. This result was also expected. So according to the results we can say that the glucose-starch separation was successful.

The samples taken from the buffer solution and the solution inside of the bag of BSA- cystein mixture was tested with Biuret reagent and Ninhydrin solution. Biuret reagent is used to observe the presence of protein (BSA) since the colour changes from blue to purple in the presence of protein. The result showed that BSA was found in both buffer and bag. This result was not expected because BSA cannot pass from the pores of membrane. So it should not be found in the buffer. The reason of that may be not to properly tying the both ends of cellulose membrane bag. Ninhydrin solution is used to observe the presence of amino acid (cystein) since the colour changes from blue to violet in the presence of amino acid. The result showed that there was cystein present in the buffer but there was not any in the bag. So we can say that cystein separation was successful.

Laboratory‎ > ‎Gel Filtration Using Sephadex v2

When a mixture of protein applied to the top of the gel filtration column, depending on the molecular exclusion limit of column material. The large protein molecules excluded from the internal volume and thus leave the column first. The smaller protein molecules equilibrate themselves between the external and internal volume and leaves the column later.
….

Laboratory‎ > ‎Heat Treatment v2

In this experiment, Major affect of enzyme activity and stability is temperature. Since enzymes are biochemical catalysts, made up at least partially of protein, they are sensitive in varying degrees to heat. Raising temperatures of the environment generally multiplies the degree of activity by the enzyme. The most dissolved protein is denatured by heat when the temperature higher than about 50oC. Heat denaturation results in protein precipitation as a result of destruction of the secondary structure and formation of random aggregates. Because all proteins are not stable when heated in aqueous solution. The precipitate enzyme is recovered by filtration or centrifugation and dried in atm. or vacuum driers. For most commercial application cost is most important than high purity and is usually unnecessary.

When the presence of contaminating enzymes or other substance will adversely affect the product and activity. If the turbidity is observed in taken tube from ice bath, it is centrifuged. Because protein is denatured due to heat treatment and so sedimentation will occur. Therefore the sedimentation part must be removed. When we look at our results it is obviously seen that increase in temperature increases the activity of enzyme up to a level. According to our result this temperature is 40oC. In 60oC the protein content is more than in 40oC but specific activity of 60oC is less. On the other hand when we compare the velocities we can say the same thing. In 30oC velocity reaches the highest value. Finally we can say that because of protein denaturation in high temperatures, enzyme activity increases up to a level with increase in temperature.

Answers of The Questions:

1.At room temperature is highest than other temperature. Because the temperature is increased as the activity is decreased. The activity is affected for the temperature. The protein structure (interaction bond) is changed.

2.State the temp. At which the purity is highest. At 40 C the purity is highest.

3.Purification helps to elimination the contaminants, which is effect the purification. The foreign material that is not wanted in product for the pure product to be best product is changed the purification. But this is more expensive, so it is usually unnecessary for most commercial application.

4.The factor that, will affect the purification. The process condition is important for purification. If the purification is not best, the enzyme activity is not best. The unpurification enzyme is not work in process. This reason is important for working and activation enzymes.

Laboratory‎ > ‎Immobilization

Purpose:

In this experiment we determined the rate of enzyme activity using free and immobilized enzyme. And our aim in this experiment, notice the difference between them.

Materials:

Distilled water, Buffer solution, Tube, Pipette, Oil, Alcohol, Water bath, Beaker, Enzyme (Candida rugosa lipase)

Procedure:

Immobilized enzyme: 0.012 g Immobilized enzyme (Candida rugosa lipase) was taken and 1 ml. olive oil was added. Then 3 ml. 25 mM KP buffer was added. This solution was placed water bath at 37 C for 30 min. After immobilized enzyme had been centrifuged, supernatant was taken. And 5 ml. alcohol (ethanol) and a few drops phenolphthalein were added. The time was recorded which is taken from the centrifuged to alcohol added. After that it was titrated with 0.05 M KOH. Finally titrated volume was recorded. Free enzyme: 0.2 ml. free enzyme was weighted then 1 ml .olive oil and 3 ml. 25 mM KP buffer were added. It was placed water bath at 37 C for 30 min. After that 5 ml ethanol and a few drops phenolphthalein were added and titrated with 0.05 M. KOH. Finally titrated volume was recorded.

Laboratory‎ > ‎Km v1

Purpose: The aim of this experiment is determining Km by using three methods which are Michaelis Menten equation, Eadie Hofstee and Lineweaver Burk plot.

Materials: Double beam spectrophotometer, test tubes, pipettes

Enzyme solution: 10 mg/ml enzyme in distilled water

Substrate solution: p-Nitrophenyl acetate in acetonitrile

Buffer: 200 mM potassium phosphate buffer, pH 7.4

Procedure: First of all, according to table below the reagents was diluted into spectrophotometer cell at each substrate concentration for each step in the table. And then the change in absorbance with time was followed by a double beam spectrophotometer for each substrate concentration in the table. However time versus absorbance graph for each substrate concentration was printed by spectrophotometer. From this graph initial rate versus substrate concentration was found. And finally from obtained data mK methods. value of the enzyme substrate reaction values was found by using three

Laboratory‎ > ‎SDS Page

The separation of macromolecules in an electric field is called electrophoresis. A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The most commonly used system is also called the Laemmli method after U.K. Laemmli, who was the first to publish a paper employing SDS-PAGE in a scientific study. SDS (also called lauryl sulfate) is an anionic detergent, meaning that when dissolved its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. In a gel of uniform density the relative migration distance of a protein (Rf, the f as a subscript) is negatively proportional to the log of its mass. If proteins of known mass are run simultaneously with the unknowns, the relationship between Rf and mass can be plotted, and the masses of unknown proteins estimated.

Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative abundance of major proteins in a sample, and to determine the distribution of proteins among fractions. The purity of protein samples can be assessed and the progress of a fractionation or purification procedure can be followed. Different staining methods can be used to detect rare proteins and to learn something about their biochemical properties. Specialized techniques such as Western blotting, two-dimensional electrophoresis, and peptide mapping can be used to detect extremely scarce gene products, to find similarities among them, and to detect and separate isoenzymes of proteins.